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纸质出版:2010
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邵会媛,杨再林,高玉洁,苗宗玉,覃凤娴,陈先春,蔡晓钟,张伶.核仁磷酸蛋白突变基因表达对NIH3T3细胞增殖和凋亡的影响[J].陆军军医大学学报(原第三军医大学学报),2010,32(08):783-786.
Shao Huiyuan,Yang Zailin,Gao Yujie,Miao Zongyu,Qin Fengxian,Chen Xianchun,Cai Xiaozhong,Zhang Ling.Nucleophosmin mutation promotes cell proliferation and suppresses apoptosis in NIH3T3 cells[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(08):783-786.
邵会媛,杨再林,高玉洁,苗宗玉,覃凤娴,陈先春,蔡晓钟,张伶.核仁磷酸蛋白突变基因表达对NIH3T3细胞增殖和凋亡的影响[J].陆军军医大学学报(原第三军医大学学报),2010,32(08):783-786. DOI:
Shao Huiyuan,Yang Zailin,Gao Yujie,Miao Zongyu,Qin Fengxian,Chen Xianchun,Cai Xiaozhong,Zhang Ling.Nucleophosmin mutation promotes cell proliferation and suppresses apoptosis in NIH3T3 cells[J].J Amry Med Univ (J Third Mil Med Univ),2010,32(08):783-786. DOI:
目的 探讨核仁磷酸蛋白(nucleophosmin
NPM)突变基因对小鼠NIH3T3成纤维细胞系体外增殖和凋亡的影响以及其分子机制。 方法 通过脂质体介导将真核表达载体pEGFP-C1-NPMc
+
转染NIH3T3细胞,G418筛选稳定表达NPM突变蛋白细胞株,RT-PCR和Western blot检测NPM突变基因和蛋白的表达。MTT、克隆形成实验检测细胞体外增殖能力;FCM检测细胞周期分布及凋亡水平的改变,并通过对周期调控因子p21及凋亡相关分子Caspase-3活性检测,初步探讨NPM突变参与细胞恶性增殖的分子机制。 结果 建立了稳定表达NPM突变基因的NIH3T3细胞株。NPM-mA实验组细胞较对照组细胞增殖能力及平板克隆形成率明显增高(P0.05);甲基纤维素集落形成实验显示各组细胞在甲基纤维素上均不能形成集落;与对照组比较
NPM-mA实验组细胞中G
1
期细胞比例(42.27±0.86)%显著减低(P0.01),S期细胞比例(43.08±0.74)%显著增加(P0.01),同时伴有p21 mRNA水平下降
稳定表达NPM突变基因的NIH3T3细胞凋亡率(1.00±0.13)%明显降低(P0.01),Caspase-3活性(0.784±0.080)下降(P0.01)。 结论 NPM突变基因可促进NIH3T3细胞体外增殖,抑制细胞凋亡发生。
Objective To investigate the effects and molecule mechanisms of nucleophosmin (NPM) mutation gene on cell proliferation and apoptosis in mouse fibroblast NIH/3T3 cells. Methods Eukaryotic expression vector pEGFP-C1-NPMc
+
was transfected into NIH3T3 cells by Lipofectamine
and the positive clones were screened by G418. Then the expression of NPM mutation gene and protein were analyzed by PCR and Western blot analysis respectively. To observe
the effect of NPM mutation on cell proliferation in vitro
MTT and colony formation assays were used. To evaluate the mechanisms of cell proliferation induced by NPM mutations
the cell cycle and apoptosis were detected by FCM
meanwhile
the cycle regulatory factor p21 was analyzed by RTPCR and the activity of Caspase-3 was detected. Results NIH3T3 cells stably expressing NPM mutation were established. The growth ability and the rate of plate colony formation of cells in experimental group were significantly higher than the control groups (P0.05). Methylcellulose colony formation assay showed that cells of all groups could not form a colony in the cell culture media. Compared with control groups
the percentage of cells in the phase of G
2
was decreased remarkably (P0.01) and that in the phase of S was increased significantly (P0.01)
and the expression of p21 mRNA was reduced notably (P0.05). The rate of apoptosis and the activity of Caspase-3 were decrease remarkably (P0.01) in experimental group. Conclusion NPM mutation gene in NIH3T3 cells can induce cell proliferation and inhibit apoptosis.
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